Reverse Genetics for Feline Morbillivirus Sequence Fidelity
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Solution Overview
Problem
There is a lack of effective methods for producing recombinant feline morbillivirus (FeMV) due to the limitations of cell passage and isolation, which can lead to viral adaptation and mutation, hindering downstream applications.
Innovation Solution
A method using reverse genetics is developed to produce recombinant FeMV by extracting RNA, generating cDNAs, amplifying genomic and antigenomic termini, purifying amplicons, sequencing, and assembling a full-length genome in a plasmid, utilizing feline CD150-expressing cells and cysteine proteases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cell passage and isolation methods are used to produce FeMV, then viral production is achieved, but viral adaptation and mutation occur leading to altered sequences
Solution Approach 1:
The patent applies preliminary action by constructing a reverse genetics system with cloned viral genome segments in plasmid vectors before virus production. The full-length FeMV cDNA is cloned into pCI-neo vectors, and viral proteins are expressed in trans to initiate virus production from cloned cDNA. This preliminary cloning and setup ensures the viral sequence remains unaltered while enabling controlled production.
Solution Approach 2:
The patent uses an intermediary approach by introducing cloned FeMV cDNA sequences into eukaryotic cells as intermediates. The viral genome is first cloned into plasmid vectors, then transfected into cells that express viral proteins in trans. This intermediary cloned DNA serves as a template for producing authentic virus without direct cell-to-cell passage, preventing mutation accumulation.
2Manufacturing precision
If reverse genetics system is used to produce recombinant FeMV, then unaltered viral sequence is obtained, but production complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the FeMV genome into separate cloned cDNA segments corresponding to individual viral genes (N, P, V, C, M, F, H, L). Each gene is cloned into separate plasmid vectors, which can be independently manipulated and then co-transfected into cells. This segmentation simplifies the management of the complex reverse genetics system while maintaining sequence fidelity.
Solution Approach 2:
The patent uses universal plasmid vectors (pCI-neo) that can accommodate multiple different FeMV cDNA inserts. The same vector system is used for cloning, expression, and transfection across all viral gene segments. This universal platform reduces the overall complexity by providing a standardized system rather than requiring unique constructs for each gene.
3Productivity
If cell passage methods are used, then viral production is achieved, but time is lost due to adaptation processes
Solution Approach 1:
The patent applies preliminary action by pre-cloning the complete FeMV genome into cDNA form and storing it in plasmid vectors before virus production is needed. When virus production is required, the cloned cDNA is directly transfected into cells with viral proteins, bypassing the time-consuming cell passage and adaptation process entirely. This preliminary cloning step enables rapid production of authentic virus.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach provides an unaltered FeMV sequence suitable for downstream applications, avoiding viral adaptation and enabling efficient production of recombinant FeMV.
Implementation Method 1
generating cDNAs from the FeMV RNA using primers that specifically hybridize to the FeMV RNA
Implementation Method 2
generating cDNA PCR amplicons from the cDNAs using primers that specifically hybridize to the cDNAs
Implementation Method 3
transfecting cells with the cloned FeMV cDNA and T7 RNA polymerase to produce recombinant FeMV
Data Source
AI summary
Disclosed are methods of producing recombinant feline morbillivirus (FeMV) using reverse genetics. In some aspects, the methods comprise: (a) extracting FeMV RNA from an isolated FeMV positive sample, (b) generating cDNAs from the FeMV RNA using primers that specifically hybridize to the FeMV RNA, (c) generating cDNA PCR amplicons from the cDNAs using primers that specifically hybridize to the cDNAs to produce cDNA PCR amplicons, (d) amplifying genomic and antigenomic termini of the FeMV RNA by rapid amplification of cDNA ends (RACE) using one or more RACE primers to produce RACE PCR amplicons, (e) purifying the cDNA PCR amplicons of step (c) and the RACE PCR amplicons of step (d) to produce purified DNA, (f) sequencing the purified DNA to produce consensus sequences, (g) assembling the consensus sequences to produce a full-length FeMV genome, and (h) assembling the full-length FeMV genome in a plasmid.